Fields as diverse as bioresearch, ecology, medicine, pharmacology, drug discovery and biohazard detection have a critical need for large-scale biomolecule affinity sensing. Affinity sensing detects the presence and/or affinity of “target” biomolecules using other “capture” biomolecules. Target molecules include DNA segments, RNA segments, protein, and small molecules and the capture molecules are often derivatives of their natural (i.e., in vivo) binding partners which are also typically DNA, RNA, and protein, but may also include small molecules having a high binding affinity. Microarray detection systems are a technology that has the potential to assess large numbers of target molecules in a relatively rapid manner, making it a useful high-throughput methodology (FIG. 1). From tens to more than a million different capturing agents are fixed in localized spots or features on the microarray substrate (e.g., glass). Typically, target molecules are obtained from a biological or environmental sample, purified, and fluorescently labeled with a dye molecule including but not limited to Cy5, Cy3, quantum dots, or biotin for subsequent labeling with streptavidin or antibodies. The prepared sample is exposed to the array. The array is then rinsed and placed in a fluorescent scanner that measures the fluorescent signal from every target molecule location.
Throughput refers to the number of target molecule spots measured in a single experiment. Each spot measures the affinity for molecules in the sample for the fixed capturing agent attached to the substrate at that position. High throughput methods are advantageous because they allow the simultaneous detection of a large number of target molecules in a single experiment.
Microarrays are actively being used today in biological research and their use is expected to expand over the next decade in the areas of medical diagnosis, drug discovery, and bio-weapons detection. Currently, DNA microarrays observing DNA-DNA interaction constitute the vast majority of microarray use, although the technology for protein-protein and protein-DNA arrays is rapidly advancing.
Despite widespread use, microarrays have their limitations. Specifically, the target molecules must be fluorescently labeled for detection. The labeling may add ambiguity and, in certain instances, precludes microarrays from many applications where affixing a label to the target molecules may not be practical or possible. Proteins, for instance, pose a particularly significant labeling challenge because attachment of a fluorescent label is likely to alter the conformation and hence the binding properties of the protein. Even DNA labeling, the most widely used and reliable microarray assay, can be unreliable because (i) the label may affect the DNA binding properties, (ii) the labeling procedure may be time consuming and costly, (iii) each DNA molecule may receive zero, one, or more than one label reducing the statistical significance of the measured result, (iv) labels may non-specifically bind to the background and cloud the signal, and/or (v) background auto-fluorescence from the substrate may cloud the fluorescent label signal. Thus, a label-free detection technology for microarray assays is preferable.
There are a number of optical label-free technologies currently under development including waveguides [Lukosz 1990], surface plasmon resonance [Brockman 2000], optical gratings [Lin 2002], and cantilevers [Zhang 2004]. None of which these technologies have yet demonstrated significant throughput. Surface plasmon resonance (SPR), for example, is available with the capability of 400 simultaneous observation sites. SPR requires the use of a metallic surface which precludes the well-established and accepted microarray chemistries developed for SiO2. Also, SPR does have the benefit of detecting binding events in real-time which enables the observer to gather kinetics information about the binding reactions. Likewise, however, standard fluorescent microarrays demand dry samples which also precludes obtaining real-time kinetics information. Thus, SPR and standard fluorescent-based microarray assays are satisfactory only for those applications in which throughput is more critical than kinetics information. There is a need for a system that may be used either for real-time measurements for kinetics information, or as a dried assay to collect only binding data.
Optical surface profilers are devices that detect small height changes across a surface using optical interference measurements. Optical surface profilers are used in many semiconductor processing labs and work by one of two principles: phase shift interferometry (PSI) or white light interferometry (WLI) (FIG. 2). PSI works by illuminating a reflecting sample with single wavelength light. The illumination beam is split so that part reflects off the sample and part reflects off a reference surface before they are recombined and imaged on a camera. The beams interfere when combined to form an interference pattern also known as an interferogram which is imaged and recorded by a camera (e.g., a CCD camera). The reference mirror position is scanned to create different path lengths for the reflected beam while the interferogram at each position is captured by the camera. When the path length between the reference reflector position and the sample surface position at a particular location is the same, or off by an integer multiple of the wavelength, the intensity at that pixel is maximum. This indicates the relative surface height at that position. This PSI method works well when the measuring relative heights shorter than the one half of the wavelength (λ/2) to avoid ambiguity in the relative measurement.
WLI is an alternative optical profiling technique that avoids the ambiguity inherent in PSI. The WLI setup and measurement procedure is essentially the same as for PSI (i.e., the light is split with part going to the sample, part going to a reference reflector, and the reflections are combined and images onto a camera). The difference between the techniques is that instead of using a single wavelength (PSI), a spectrally broad illumination source is used. With a spectrally broad source, the two combined beams will only interfere constructively when the path length for either reflection is the same without the integer multiple of the wavelength caveat of the PSI method. These existing optical profiling methods in the semiconductor field, however, lack sensitivity for biosensing applications where low-index biomaterial is binding to a low-index glass surface.
Optical interferometric methods have been more recently developed specifically for biosensing applications [Pichler 1996; Moiseev 2006]. One such method is spectral interference (SI). The sample used in SI consists of target molecules on a semitransparent layer. Light reflecting from both the top and bottom surfaces of the semitransparent layer with the biomaterial interferes in the reflected beam (FIG. 3). The spectrum of the combined reflections from the top and bottom layer interface is used to determine the layer thickness and hence molecule binding to the substrate surface. Some wavelengths will experience constructive interference while other wavelengths will experience destructive interference based on the thickness of the semitransparent layer. There are two primary reflections, one from the top surface and one from the bottom surface with less significant higher order reflections that make multiple passes through the semitransparent layer and contribute less to the signal. While these secondary reflections contribute less, they do improve the measurement by helping distinguish the wavelengths experiencing constructive interference from wavelengths experiencing destructive interference. The combined reflected beam is measured by a spectrometer. Wavelengths where the interference is destructive are attenuated. The spectrum is used to determine at which wavelengths the interference is destructive and constructive, and hence the apparent thickness of the semitransparent layer, for which bound biomaterial increases the apparent thickness. Thus, knowing the initial thickness of the semitransparent layer allows a calculation of the height of the biomaterial on the surface. The disadvantage of this method for use as a biosensor is that the method is performed by a spectrometer and can be applied to only one location at a time. It is difficult to measure the spectrum of spectrally broad light at many nearby locations simultaneously, meaning that the technique is not useful for the high resolution imaging necessary to read modern microarrays.
The present invention solves many of the problems of the prior art, including providing a real-time, label-free microarray system suitable for high throughput screening. Additionally, the invention may be adapted to take advantage of the well-established chemistry developed for attaching capture molecules to SiO2-based microarray substrates.